Published June 1, 1966
A F F I N I T Y OF A N I M A L CELL
N U C L E O L I FOR N O R M A L S E R U M
Preliminary Characterization of Serum and Cell Components
JOHN
C. M A I S E L
and R A L P H
I. L Y T L E
From the United States Naval Medical Research Unit No. 4, United States Naval Hospital, Great
Lakes, Illinois. Dr. Maisel's pt~esent address is the Department of Pathology, University of Colorado
Medical Center, Denver
ABSTRACT
There is no published concept of nucleolar composition and function to account for the observation
that " n o r m a l " sera of a wide variety of animal
species has affinity in mass cell cultures for nucleoli of living ceils of the same or different animal
species (1). This nucleolus-serum interaction is
demonstrated in two steps: first, living cell-culture
monolayers are incubated with serum not made
hyperimmune to any antigen; second, after washing the monolayer and fixing it in acetone, it is
incubated in the presence of calcium ions with an
overlay of fluorescein-labeled serum proteins.
Finally, in monolayers washed a second time and
viewed by fluorescence microscopy, intense nucleolar fluorescence is observed (I).
It should be emphasized that the labeled proteins used in the second overlay, unlike those used
in fluorescent antibody staining (2), are not
g a m m a globulins derived from animals specifically
hyperimmunized against the serum used in the
first overlay. Apparently, in the presence of calcium ions, other fluorescein-conjugated serum proteins are bound by the serum-modified nucleolus
(1). The mechanism and specificity of the second
step have not been elucidated; nor has it been
quantitated.
It is of more immediate interest to understand
the events and significance of the first step, i.e., the
modification of nucleoli by serum. This paper records the effects upon the phenomenon of modifying or fractionating serum in a number of ways, or
of altering nucleolar composition. Nucleolar protein appears to be the cell component with affinity
for serum. One fraction of serum interacting with
nueleoli is a n a l p h a globulin rich in glycoprotein.
This same fraction in addition can be substituted
for whole serum in supporting, over a 1 wk period,
461
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Nucleoli of animal cells cultured in vitro arc modified by a component of " n o n i m m u n e "
animal serum. Modified nucleoli bind fluorcsccin-conjugated nonimmune serum proteins,
as shown by calcium ion-dependent fluoresccncc. Analysis of serum indicates that the
nucleolar-binding component is a globulin, with an clectrophoretic mobility in the same
region as the slow alpha-1 component in p H 8.6 Veronal buffer. T h e component has a low
sedimentation constant (2.4S), and appears to contain glycoprotein with relatively high
sialic acid content (8.5 %) ; thc latter moiety may bc essential to reaction with nucleoli. The
nucleolar component reacting with this alpha globulin fraction appears to be a histonelike
basic protein. Primary cultures of animal cells have been supported for 1 wk through attachmcnt, spreading, and outgrowth from colonies to confluent monolaycrs in medium
containing a nucleolar-reactive serum fraction as the only protein supplement.
Published June 1, 1966
the outgrowth of a confluent cell monolayer from
primary explants of primate cells.
MATERIALS
AND
METHODS
Serum Modification (Table I)
Serum Fractionation (Table II)
Fresh h u m a n serum was fractionated by: the cold
alcohol procedure of C o h n (method 6) (4); ultra
centrifuge flotation (5); the perchloric acid-phosphotungstic acid fractional precipitation sequence devised by Winzler for orosomucoid (6); continuousflow, paper-curtain electrophoresis (7); or precipitation by addition of an equal volume of a mixture of
alcohol and ether in 3:1 ratio. Precipitates were redissolved in 0.14 M NaC1. O n e fraction recovered by
curtain electrophoresis and exhibiting nucleolar
affinity was refractionated on the curtain and the
products were analyzed further.
Analysis of Serum Fractions
SERUM
ELECTROPHORESIS
(TABLE
III,
Electrophoretic analysis of h u m a n
serum fractions was carried out on paper strips in a
S h a n d o n tank with 0.075 ionic strength Veronal
buffer at p H 8.6 for 15 hr at 5 ma.
CARBOHYDRATE
ANALYSIS : Carbohydrate
analysis of the "nucleolar-reactive" serum fraction
isolated by curtain electrophoresis was kindly perP A R T A) :
462
Histochemical Analysis of Nucleoli (Table
IV)
Acridine orange fluorescent staining as described
by Bertalanffy (8) was used to evaluate the effectiveness of enzymatic removal of nucleic acids from cell
monolayers. Extraction with various inorganic electrolyte solutions followed a scheme evolved by Duryee
(9). Histone extraction with nonisotonic saline or
cold mineral acid was according to Dounce (10).
Cells treated by either m e t h o d were evaluated for
binding with a known nucleolar-reactive serum by
the nucleolar fluorescence test described below.
Cell Cultures
Fresh adult Macaca mulatta renal cortical cells were
trypsin-dispersed according to Youngner (l l) and
resuspended at a concentration of 200,000 cells per
milliliter in " m e d i u m 199" supplemented with 10%
calf serum and adjusted to p H 7.2 with N a H C O v
M e d i u m 199 is a "defined m e d i u m " containing a
balanced salt mixture, essential and nonessential
amino acids, vitamins, and nucleosides (12), but
requiring a protein supplement for optimal growth
of most cells. This introduces undefined factors into
the final preparation. Two ml of cell suspensions was
delivered into 80-mm Leighton tubes containing
11 X 44 m m cover slips. T h e cultures were incubated at 37 ° C in a humidified 5 % COs atmosphere.
W h e n the monolayers were confluent, usually on the
4th day after explanting, the growth m e d i u m was
removed and the cover slips were washed with phosphate-buffered saline (PBS, 0.15 M NaC1, 0.01 M
POa, p H 7.4), in preparation for fluorescent staining
(see below). Replicate cultures planted in screw cap
tubes were harvested daily for cell counting. Vigorous washing prior to trypsin detachment ensured
that only cells attached to glass would be enumerated.
T h e dispersed cells were mixed with 10-5% trypan
blue to exclude counting nonviable cells in the hemocytometer.
ThE JOURNAL OF CELL BIOLOGY " VOLUME ~9, 1966 "
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Aliquots of fresh h u m a n serum were each treated
by one of the following procedures: (a) Heat inactivation, adsorption on zymosan, or mild acid or
alkaline hydrolysis, following the methods of K a b a t
and Meyer, was applied to serum aliquots for preparation of reagents deficient in one of the fractions of
serum complement (R-l, R-2, R-3, or R-4) (3).
(b) Enzymatic digestion of individual allquots was
accomplished with trypsin, Vibrio cholera crude filtrate (24 hr broth culture with ability to liquify
gelatin), or influenza A-PR8 virus neuraminidase,
as infective virus (for details, see Table I). (c) Dialysis was carried out separately on each of three 5-ml
aliquots of serum in Visking tubing at 4 ° C for 15
hr, against either 3 changes of constantly stirred,
200 ml volumes of 0.14 M NaCI or distilled H20,
or against one 200 ml volume of 30% polyvinylpyrrolidone (PVP). (d) Adsorption on suspensions
of kaolin was as described in Table I. (e) Reaction
with periodate was as detailed in Table I. T h e products of these treatments were tested for nucleolar
affinity as described below. I n some cases quantitative comparison was attempted by testing serial
dilutions of the product. Before the serum products
were tested, they were concentrated to the original
volume by dialysis against PVP.
formed by Dr. R. J. Winzler (Department of Physiological Chemistry, University of Illinois, Chicago),
according to published methods (6).
SEDIMENTATION
VELOCITY
:
Analytical ultracentrifugation, of a nucleolar-reactive serum fraction isolated by curtain electrophoresis, using a
"model E" analytical ultracentrifuge (Spinco Div.,
Beckman Instruments, Inc., Palo Alto, California)
was as described by Schachman (5). T h e sample,
dissolved in p H 8.6, 0.02 ionic strength Veronal
buffer, contained 3 mg of protein (Lowry) per milliliter. Determinations were made at 16-sec intervals
after attainment of 59,780 RPM.
Published June 1, 1966
TABLE I
Nucleolar Reactivity* of Variously Modified Human Serum
Method of degradation
Nucleolar reactivity at seTurn
dilution tested§
Product:~
D i a l y s i s (5 m l s e r u m in V i s k i n g t u b i n g ,
3 c h a n g e s o f 200 m l v o l u m e s )
(H20)
15 h r at 4 ° C
M-supernate
E-precipitate
Dialyzed serum
Concentrated serum
P r e s e n t at 1:1 (not t i t r a t e d
H or " i n a c t i v a t e d " s e r u m
P r e s e n t t h r o u g h 1:20
P r e s e n t t h r o u g h 1:20
T r y p s i n (1% in 5 0 % s e r u m ; 1 h r at
37 °C)
Digested serum
Present through 1: 1 only
V. cholera c r u d e filtrate (1:20 in 2 0 %
Digested serum
A b s e n t at 1:5 (not t i t r a t e d )
P e r i o d a t e (0.5 m g K 1 0 4 p e r m l s e r u m ,
1 h r at 22°C, d a y l i g h t )
P r o d u c t s of s e r u m
A b s e n t at 1:5 (not t i t r a t e d )
H y d r o l y s i s (0.15 M final N H 4 O H or
HC1, 1 h r at 22°C); N
Hydrolyzed serum products; R - 4
A b s e n t at 1:5 (not t i t r a t e d )
N e u r a m i n i d a s e ( I n f l u e n z a A-PRB-34
virus, 256 H A u n i t s final, 1 h r at
37 °C)
Digested serum products
A b s e n t at 1:2 (not t i t r a t e d )
Absorption
Z y m o s a n (type A : 1.35 m g / m l ; Z)
K a o l i n (12.5% final, 1 h r at 37°C)
R - 3 or s e r u m a b s o r b e d of
o n e or m o r e c o m p o n e n t s
P r e s e n t at 1:2 (not t i t r a t e d )
P r e s e n t t h r o u g h 1:20
E + H (see above)
R-I
P r e s e n t t h r o u g h 1:20
M + H (see a b o v e )
R-2
P r e s e n t t h r o u g h 1:20
(NaC1, 0.14 M)
( P V P , 30%)
"
"
P r e s e n t at 1:1 (not t i t r a t e d
P r e s e n t at 1:1 (not t i t r a t e d
Heat
(20 m i n at 56°C;
1 h r at 6 0 ° C )
s e r u m , 2 h r at 37°C)
Growth Supporting Effects
BRIEF CONTACT OF CELLS WITH
WHOLE SERV~
Fresh trypsin-dispersed m o n k e y kidney cells were
washed three times in PBS to r e m o v e trypsin a n d
s e d i m e n t e d at 1000 RPM for 5 rain in a clinical centrifuge; the fluid was decanted. T h r e e series of cell
cultures were established, one test series a n d two
control series (Fig. 2). T w o - t e n t h s m l packed v o l u m e
of "test" cells was r e s u s p e n d e d in 0.5 m l of fresh
whole h u m a n s e r u m (type O, R h positive), a n d t h e
m i x t u r e i n c u b a t e d for 1 h r at 37 ° C. T h e cells were
resedimented; t h e s e r u m was decanted a n d saved for
further evaluation (see below, a n d Fig. 2). T h e cells
were w a s h e d by two cycles of alternate low speed
s e d i m e n t a t i o n a n d resuspension in 5 ml v o l u m e s of
J. C. MAIS~.L ANn R. I. LYTLE
Affinity of Cell Nucleolifor Serum
463
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* N u c l e o l a r r e a c t i v i t y is d e f i n e d q u a l i t a t i v e l y as t h e a b i l i t y o f s e r u m or s e r u m f r a c t i o n to p r o d u c e n u c l e o lar fluorescence w i t h cell m o n o l a y e r s in t h e c a l c i u m i o n - d e p e n d e n t , n o n i m m u n e f l u o r e s c e n t t e c h n i q u e
d e s c r i b e d in t h e text.
:~ E, M , H , N , Z a n d R-1 t h r o u g h R - 4 refer to m o d i f i e d s e r u m deficient in o n e or m o r e e l e m e n t s of t h e
c o m p l e m e n t s y s t e m a c c o r d i n g to K a b a t a n d M a y e r (3).
§ I n s o m e cases, p r o d u c t s were r e c o n c e n t r a t e d to o r i g i n a l s e r u m v o l u m e s a n d q u a n t i t a t i v e c o m p a r i s o n s
attempted by serological titration.
Published June 1, 1966
m e d i u m 199 u n s u p p l e m e n t e d with s e r u m or a n y
other protein. Replicate 2 m l v o l u m e s of cell suspension containing 160,000 ceils in fresh m e d i u m 199
were placed in Leighton tubes containing cover slips,
or in stationary screw cap test tubes. I n t h e first
control series, the trypsin-dispersed, PBS-washed
ceils were not i n c u b a t e d with s e r u m ; after two washings in m e d i u m 199, the cells were resuspended in
fresh m e d i u m 199 a n d cultures established as indicated above. I n the second control series, cells not
i n c u b a t e d with s e r u m b u t w a s h e d in m e d i u m 199
were resuspended in fresh m e d i u m 199 s u p p l e m e n t e d
with 10% calf serum, a n d cultures established as
indicated above. Cell cultures in all three series of
L e i g h t o n a n d screw- c a p tubes were c o m p a r e d daily
for o u t g r o w t h of cell monolayers. I n addition, o n e
screw cap t u b e culture f r o m each series was harvested daily for m e a s u r e m e n t of increase in cell
number.
T h e "test" s e r u m decanted after a 1 hr i n c u b a t i o n
with the test cells was serially diluted in twofold
progression b e g i n n i n g with a n initial dilution of
1:1.25 in PBS. S e r u m i n c u b a t e d for 1 hr at 3 7 ° C
w i t h o u t cell suspension was serially diluted as a control (not s h o w n in Fig. 2; c o m p a r e with c o l u m n I).
Both the test a n d control s e r u m dilutions were eval-
T A B L E II
Biochemical Fractionation of Nucleolar-Reactive* Component from Human Serum
Fraction
Method
Reference
Name
Main content
Protein
concentration
Nucleolar
reactivity
mg/ml
C o h n ( m e t h o d 6)
10
Absent
Absent
Present
Absent
Absent
Absent
Si 0-12
12-30
20 a n d g r e a t e r
Lipoproteins
Lipoproteins
Lipoproteins
20
Present
Present
Present
Supernate
P r e c i p i t a t e redissolved in 0.14 M
NaC1
T o t a l lipids
Serum protein
-10
Absent
Present
Precipitate A
Supernate A
Protein
Soluble seromucoid
10
10
Absent
Absent
Precipitate B
Supernate B
Mucoprotein
--
10
--
Absent
Absent
C o l l e c t i o n t u b e s 13
to 15
All o t h e r t u b e s
Alpha globulins
0.5
Present
Other proteins
0.5
Absent
Collection t u b e s 16
to 20
All o t h e r t u b e s
Slow alpha-1 g l o b u l i n s
0.3
Present
Other alpha globulins
0.3
Absent
II
III-1
IV-I
V
VI
Ultracentrifuge flotation
(15 h r at 30 000 g in
1.13 sp gr NaC1)
Precipitation I
A l c o h o l - e t h e r 3:1 w i t h l
volume serum, 4°C
P r e c i p i t a t i o n II
A 0.6 ~t H C 1 0 4
B
P h o s p h o t u n g s t i c acid
to s u p e r n a t e A
Curtain electrophoresis
exp. A, initial r u n
(whole s e r u m )
r e f r a c t i o n a t i o n r u n (tubes
13 to 15 above)
* See T a b l e I.
464
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Fibrinogen
Gamma globulin
Beta globulins
Alpha globulins
Albumin
Salts, n o n p r o t e i n , alpha globulins, prealbumin
I
Published June 1, 1966
TABLE III
Properties of Curtain Electrophoresis Fractions of Serum
Part A
C o m p a r i s o n of e l e c t r o p h o r e t i c m i g r a t i o n a n d n u e l e o l a r reactivity* of fractions of h u m a n
serum
Relative values$, per cent
Serum fraction
Part B
0
A
2
a- 1
a-intermediate
a-2
B
3'
Nucleolar
reactivity*
15
60
20
Present
100
Absent
Absent
Present
Absent
Absent
serum
3
N o t tested
100
Combined alpha's, 100
100
100
25
100
100
100
100
100
75
100
Absent
Present
Present
Present
Present
Present
Absent
C o m p a r i s o n of g r o w t h - s u p p o r t i n g p r o p e r t i e s a n d nucleolar reactivity* of fractions of h u m a n
serum
Curtain electrophoresis serum fraction
Experiment A
Refractionation run, tube
18
Experiment B
I n i t i a l r u n , tubes
11-16
17-18
19
20-23
24
25-30
Outgrowth of primary explants of monkey kidney cells in medium
199 supplemented with serum fraction instead of whole serum§
Nucleolarreactivity*
C o n f l u e n t m o n o l a y e r in 5 to 7 days
Present
Small islands of cells after 7 days
Confluent m o n o l a y e r s in 5 to 7 days
Cells died after 1 to 2 days
C o n f l u e n t m o n o l a y e r s in 5 to 7 days
C o n f l u e n t m o n o l a y e r s in 5 to 7 days
Small islands of cells after 7 days
Absent
Present
Absent
Absent
Present
Absent
* See T a b l e I.
:~ R e l a t i v e a m o u n t s as d e t e r m i n e d by p a p e r strip electrophoresis e m p l o y i n g 0.075 ionic s t r e n g t h Verona1
buffer, p H 8.6, S h a n d o n tank, a n d 5 m a for 15 hr.
§ See M a t e r i a l s a n d M e t h o d s for details of cell cultures.
uated for ability to mediate "nucleolar fluorescence"
in the test to be described below. Results for the test
s e r u m are presented in Fig. 2, c o l u m n 1.
Finally, cell monolayers developing o n cover slips
from the test cells were harvested on the 7th day after
planting. T h e y were washed in PBS, fixed in acetone,
a n d prepared for fluorescence microscopy as will be
described below, with the i m p o r t a n t exception that
the usual first overlay with " n o n i m m u n e " s e r u m
was omitted at this time. Similar preparations were
m a d e with control cells g r o w n in 10% calf serum. T h e
monolayers were scored for presence or absence of
J. C. MAISEL ASD R. I. LYTLE Affinity of Cell Nucleoli for Serum
465
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Cohn III-I
C u r t a i n electrophoresis
E x p e r i m e n t A initial r u n
with whole serum tubes
6 to 9
10 to 12
13 to 15
16 to 20
21 to 24
R e f r a c t i o n r u n w i t h t u b e s 13 to
15 from a b o v e t u b e
15
16
17
18
19
20
21
Prealbumin
Published June 1, 1966
T A B L E IV
S e r u m R e a c t i v i t y * of E x t r a c t e d R h e s u s R e n a l E p i t h e l i a l Cells 3~
Nuclcolar fluorcscencc*
Treatment
Nuclease§ digestion
Conditions
5 h r at 37°C
Buffer only
RNase
DNase
Both
E x t r a c t i o n w i t h electrolyte s o l u t i o n s
S a l i n e (NaC1)
0.14 M (isotonic)
2.0 M ( h y p e r t o n i c )
0.05 M ( h y p o t o n i c )
KC1, 0.1 M
Acridine orange staining~
N u c l e o l i pink
Nucleoli green
Nucleoli orange
Nucleoli dark
Unfixed cells~:
Absent
Absent
Absent
Absent
Fixed cetls~:
Present
Present
Present
Present
Phase-contrast microsc o p y of n u c l e o l u s
1
1
1
1
hr
hr
hr
hr
at
at
at
at
22°C
22°C
22°C
22°C
Intact morphology
Blurred outline
Unchanged
Condensed
1
1
1
1
hr
hr
hr
hr
at
at
at
at
4°C
22°C
22°C
22°C
Unchanged
Swollen, e v e r t e d
Unchanged
Unchanged
Intense, compact
Absent
Absent
Present, s c a t t e r e d
in n u c l e a r loci
Intense, compact
Absent
Absent
Absent
Not
Not
Not
Not
tested
tested
tested
tested
Not
Not
Not
Not
tested
tested
tested
tested
M
* S e r u m r e a c t i v i t y is d e f i n e d q u a l i t a t i v e l y as t h e ability of cell monolayers to p r o d u c e n u c l e o l a r fluoresc e n c e w i t h s e r u m in t h e c a l c i u m i o n - d e p e n d e n t , n o n i m m u n e f l u o r e s c e n t t e c h n i q u e d e s c r i b e d in t h e text.
:~ Cell m o n o l a y e r s p r e p a r e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s were e x t r a c t e d before or after fixation,
as listed above. R e s u l t s w i t h a c r i d i n e o r a n g e s t a i n i n g were s i m i l a r w i t h fixed or u n f i x e d cells a n d w e r e
n o t a l t e r e d b y e x t r a c t i o n w i t h e l e c t r o l y t e solutions.
§ Bovine pancreatic deoxyribonuclease (DNase) and ribonuclease (RNase) (Worthington Corporation)
as 0.05 m g / m l s o l u t i o n s in p H 7.45, 0.05 M p h o s p h a t e b u f f e r c o n t a i n i n g 0.01 M MgCI~.
nucleolar fluorescence (see below). Results are given
in Fig. 2, c o l u m n s 2 a n d 3.
PROLONGED CONTACT OF CELLS WITH
S E R U M FRACTIONS
Fresh trypsin-dispersed m o n k e y kidney cells were
s u s p e n d e d in m e d i u m 199 at a concentration of 200,000 cells per milliliter. T w o ml replicate v o l u m e s were
delivered into Leighton tubes containing cover slips.
T o each t u b e was a d d e d a protein s u p p l e m e n t that
consisted of sterile whole s e r u m , o1" one of twenty
s e r u m fractions (tubes 11 to 30) derived from a n
"initial r u n " of continuous-flow, p a p e r - c u r t a i n electrophoresis (Table III, part B). T h e s e fractions were
bacteriologically sterilized by filtration. T h e a m o u n t
of s e r u m substitute in the m e d i u m of each t u b e was
adjusted to 6.8 m g of protein per milliliter (Lowry
m e t h o d ) ; this is the a p p r o x i m a t e a m o u n t of protein
present in the 10% s e r u m m e d i u m . T h e culture
robes were e x a m i n e d daily for 7 days a n d observations m a d e of cell a t t a c h m e n t , spreading, colony
formation, a n d formation of confluent monolayers.
466
Cell counts were not made. Nucleolar reactivity or
the lack of it was d e t e r m i n e d for each initial r u n
" c u r t a i n " s e r u m fraction (tubes 11 to 30) employed.
In similar fashion, refractionation r u n c u r t a i n s e r u m
fraction 18 (Tables II a n d III, part A) was used as
s e r u m substitute in cell g r o w t h m e d i u m .
Nucleolar Fluorescence
The
"indirect"
fluorescent-antibody staining
m e t h o d of Liu, Eaton, a n d Heyl (2) was modified
as follows: (a) Cell monolayers on cover slips were
r e m o v e d f r o m the g r o w t h m e d i u m , washed in three
changes of r o o m t e m p e r a t u r e PBS, a n d drained. (b)
T h e still viable cells were overlaid with 0.25 m l of
s e r u m a n d the preparation i n c u b a t e d at 37 ° C for
30 rain, in a humidified atmosphere. T h e s e r u m m a y
be a n y fresh s e r u m obtained f r o m n o n i m m u n i z e d
animals or h u m a n s ( n o n i m m u n e or n o r m a l serum).
(c) T h e m o n o l a y e r was washed in three changes of
PBS containing c a l c i u m ions, such as Dulbecco a n d
Vogt's PBS, p H 7.4 (13), containing 0.1 g m of CaC12
per liter; t h e MgC12 m a y be omitted. (d) T h e w a s h e d
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HC1, 0.2 u
HaBO4 , 0.1 M
CaCI2, 0.005 M
MgC12, 1 M or 0.001
Other observations
Published June 1, 1966
monolayer was drained, and fixed in absolute acetone
for l0 rain at 4°C. (e) Before conjugation of normal
serum, g a m m a globulin was removed by electrophoresis convection (22). Conjugation of serum proteins with fluorescein isothiocyanate was as used by
Liu et al. (2). The fixed monolayer was rehydrated
in calcium-containing PBS, drained, and overlaid
with 0.25 ml of fluorescein-eonjugated normal serum
proteins, and reincubated as before. (f) After the
second incubation, the monolayer was washed in
three changes of PBS containing calcium, and the
cover slip mounted (on a 1 X 3 in. microscope slide,
1 m m thick) in buffered glycerol (glycerol, United
States Pharmacopoiea, 9 parts, calcium-containing
PBS, p H 7.4, 1 part). (g) The preparation was
viewed as described (2).
RESULTS
Nucleolar Fluorescence
FIGURE 1 Nucleolar fluorescence with normal serum. Monolayer culture of H.Ep.-~, epithelial-like
human tumor cells prepared as described in text. Near-ultraviolet, dark-field illumination. )< 400. From
Maisel, J. C., J. Lab. and Clin. Med., 196~, 60, 857.
J. C. MAISEL AND R. I. LYTLE A~nity of Cell Nucleolifor Serum
467
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Modification of nucleoli d u r i n g the first incubation with whole n o n i m m u n e serum or serum comp o n e n t can be d e m o n s t r a t e d in our system only
after fixation of the cells a n d incubation with a
second overlay of fluorescein-conjugated serum
proteins in the presence of calcium ions (1). S e r u m
or its c o m p o n e n t s are termed nucleolar reactive if
in the nucleoli of every cell in the treated m o n o layer one sees green-yellow fluorescence t h a t is
more intense t h a n t h a t seen in the s u r r o u n d i n g
nonnucleolar nucleoplasm or cytoplasm (Fig. 1).
W i t h o u t the first i n c u b a t i o n with serum or reactive
component, nucleolar fluorescence other t h a n faint
green-blue autofluorescence of unstained cells or
tissue is not seen after " n o n i m m u n e conjugate" is
applied (1, 2). S e r u m or its c o m p o n e n t s are termed
nonreactive w h e n their e m p l o y m e n t in the first
step of the two-step procedure produces results no
different from those o b t a i n e d w h e n they are
omitted. (As used here, the t e r m reactive has no
specific meaning, since the chemistry of the modification remains unknown.) I t is only a p p a r e n t
that, after exposure of cells to serum or certain
serum components, nucleoli are able to bind, in
the presence of calcium ions, one or more unidentified serum proteins labeled with fluorescein.
Published June 1, 1966
Serum Modification
Serum Fractions
To identify the nucleolar-reactive serum component or components, h u m a n serum fractions
prepared as outlined in Table II were applied to
cell monolayers as described, and the preparations
observed for presence or absence of nucleolar
fluorescence. Fractions associated with nucleolar
fluorescence were Cohn fraction I I I - I , all three
flotation classes of "lipoproteins," alcohol-ether
precipitated proteins, and curtain electrophoresis
collection tubes 13 to 15 from the initial run.
Electrophoretic analysis of these reactive fractions
is presented in Table III, part A, and indicates that
alpha globulins are the only components common
to all the reactive fractions tested. The pooled contents of curtain electrophoresis collection tubes 13
to 15 from the initial run were refractionated.
Nucleolar reactivity was now found in collection
tubes 16 to 20. Electrophoretic analysis of these
"refractionation r u n " products also is presented in
Table I I I , part A, and it appears that nucleolar
reactivity is associated with a component migrating
in the alpha-I globulin region, as determined by
comparison with migration patterns of whole
serum run simultaneously.
468
ThE
JOURNAL OF C E L L BIOLOGY • VOLUME
29,
Chemical Analysis
T h e protein and carbohydrate contents of the
nuclear reactive fraction in second run curtain
electrophoresis collection tube 18 were determined
by Dr. Winzler to be: 6.8 mg of total protein per
milliliter (Lowry); hexose (anthrone) 6.9%;
hexosamine (Elson-Morgan) 2.2 %; and sialic acid
(Ehrlich) 8.5%. Thus the material contains approximately 15% carbohydrate, with high content of sialic acid.
SEDIMENTATION
V E L O C I T Y : An estimate of
the number of molecular species in the same material is provided in the demonstration of two peaks
in the analytical ultracentrifuge: approximately
95% of the material has a s20,w value of 2.4,
whereas the remainder has a value of 5.25. Although other parameters needed for accurate
calculation have not been determined, these data
suggest that the material in the main peak should
have an average molecular weight of the order of
40,000 to 50,000. The material in each of the peaks
posssesses nucleolar reactivity. H o w many proteins are present in either peak is not known.
Nucleolar Component
The nature of the component of the nucleolus
essential to nucleolar absorption of the serum component was investigated by histochemical methods,
as outlined in Table IV. A distinction was m a d e
between nucleolar nucleic acids and nucleolar
proteins. It is clear that, after nuclease digestion
had removed all nucleolar nucleic acid detectable
by acridine orange fluorescence from fixed cells,
nucleolar nonimmune fluorescence was still
demonstrable. Conversely, unfixed cells extracted
with hypotonic or hypertonic saline retained nucleic acids as interpreted by acridine orange
staining, but they lost their nucleolar affinity for
serum as interpreted by n o n i m m u n e fluorescent
1966
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The characteristics of the human serum component responsible for the reaction with nucleoli
were each investigated by modifying aliquots of
whole serum in one of a variety of ways. Products
of serum aliquots individually treated as listed in
Table I were applied to cell monolayers as described, and the preparations were observed for
presence or absence of nucleolar fluorescence. The
data indicated that the nucleolar reactive component of serum is a nondialyzable, heat-stable
substance not adsorbed on kaolin or zymosan. Its
activity is reduced following treatment with
trypsin or crude Vibrio cholera filtrate. Nucleolar
affinity is lost after alkaline or acid hydrolysis, or
periodate treatment of serum. Affinity of serum or
its fractions for nucleoli is reduced at least fourfold
by infective influenza virus, a fact which suggests
that neuraminidase activity split off a sialic acidreactive group of a serum protein (6). As shown in
Fig. l, if the serum tested for nucleolar affinity
previously had been incubated briefly with live
animal cells, its nucleolar reactivity was greatly
reduced. This is an indication of either removal of
the component from serum or its inactivation, by
living cells under in vitro conditions.
An estimate of the degree of concentration of
nucleolar-reactivity in the refractionated alpha
globulin material (tube 18, Table I I I , part A)
compared to whole serum was made as follows:
whole serum was not reactive beyond a 1:90
dilution, or 3.4 mg of total protein per milliliter.
The curtain electrophoresis refractionation tube 18
material, active at the tested concentration of 0.3
mg of protein per milliliter, represents a tenfold
concentration of specific effect. Whether further
concentration of effect could be achieved has not
been determined.
Published June 1, 1966
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J. C. MAISEL AND R. I. LYTLE A~nity of Cell Nucleoli for Serum
469
Published June 1, 1966
staining. Isotonic saline appeared to provide
optimal stabilization of the nucleolar protein
material. Loss of the serum-reactive material from
nucleoli of extracted cells did not always correlate
with morphological changes under phase-contrast
microscopy. For example, after KC1 treatment,
nucleoli observed by phase-contrast microscopy
appeared condensed. However, the fluorescent
material formerly associated with the nucleolus
was absent. Instead, the nucleoplasm contained
m a n y small fluorescent loci.
Growth Supporting Effects
BRIEF CONTACT OF CELLS WITH
WHOLE S E R ~
470
PROLONGED CONTACT OF CELLS WITH
SERUIVI FRACTIONS
The growth-supporting effects of twenty serum
fractions obtained from an initial run of curtain
clectrophoresis were compared with the effects of
whole serum. Confluent monolayers formed only
when whole serum, or the fractions of whole serum
collected in tubes 17 and 18, and 20 to 24 (Table
I I I , part B), were added to the culture. There was
a limited cell survival with all other fractions except for the fraction in tube 19. Nuclcolar fluorescence, however, was mediated only by the material in tubes 17, 18, and 24. T h e material in tube
19 possessed no activities. This experiment is
important because fractions from two sharply
divided electrophoretic classes of proteins supported outgrowth of confluent monolayers. In
the "faster" migrating group of five fractions (20
to 24), only the "fastest" migrating fraction, 24,
also possessed detectable nucleolar reactivity. T h e
material in four "intermediate" tubes (20 to 23)
supported monolayer outgrowth but did not
mediate nuclcolar fluorescence. In contrast, all
fractions mediating nucleolar fluorescence also
supported monolayer outgrowth.
Electrophorefic analysis of these initial run
"curtain" fractions was not made. Information
from other curtain clectrophoretically separated
serum suggests that the two active fraction groupings may represent overlapping alpha and beta
globulins (tubes 17 and 18) and overlapping alpha
globulins and albumin (tubes 20 to 24) (7). T h e
"slow alpha-1 globulin" (curtain electrophoresis
refractionation tube 18: see Tables II and I I I ,
part A) was also tested for growth-supporting
effects. The chemical and sedimentation data are
given above. This material, in addition to mediating nuclcolar fluorescence, supported outgrowth
of confluent cell monolayers in 5 to 7 days (Table
I I I , part B). The actual increase in cell number
was not determined for the monolaycrs supported
by the seven initial curtain fractions or by the one
curtain-refractionated specimen. We infer from
observations of other cultures that sequential
ThE JOURNAL OF CELL BIOLOGY " VOLUME29, 1966
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T h e test cells incubated briefly with whole
serum, after washing and suspension in medium
199 unsupplemented by serum or other protein
(Fig. 2, column 2), exhibited a "plating efficiency" of about 0.5 % as determined by a count
of 3.7 )< 103 attached viable cells 24 hr later.
Throughout the next 6 days, the cell count in
replicate cultures increased slowly until it reached
a count of 3.5 X t04 viable cells on the 7th day.
Observation of the cultured cells revealed that
small islands of cells present on the 3rd day became
progressively larger until confluent monolayers
had developed by the 7th (Fig. 2). These results
were similar to those obtained with the control
cells grown in medium supplemented with calf
serum (column 3), as well as those reported by
Youngner (11). In contrast, control cells which
were not incubated with serum before plating and
which were grown in the absence of serum supplemented medium died within 94 hr (column 4).
The 7th day cover slip monolayer arising from
serum-incubated test cells, when prepared for
fluorescence microscopy as specified above, exhibited brilliant nucleolar fluorescence in every
cell (column 2). This occurred even though at this
time we omitted the first overlay with nonimmune
serum. O n the other hand, control cells grown in
10% calf serum did not exhibit nucleolar fluorescence after similar preparation (column 3).
It appears on the basis of these data that the
technique of demonstrating nucleolar fluorescence
was successfully modified, in that the first exposure
of cells to serum was moved ahead in time to the
point when the cell cultures were established. Apparently, sufficient serum was transferred or serum
effect was imposed initially, so that nucleoli pres-
ent in monolayer cells after 7 days still bound the
conjugated proteins. At the same time, the usual
method of growing cells in serum-supplemented
medium was successfully modified, in that contact of cells with serum was brief and occurred
prior to establishment of cultures in defined
medium without serum.
Published June 1, 1966
development of cell islands and cell monolayers
indicates that cell growth is occurring, with an
increase in cell number as well as in cell size.
DISCUSSION
J. C. M ~ s ~ A~TDR. I. :LYTLE Affinity of Cell Nucleoli for Serum
471
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The data prcscntcd suggest two intcrprctations.
In the first concept, one or more alpha globulins
is adsorbed by thc nucleolus of living cells, and,
after fixation of the cells and in the prcscncc of
calcium ions, the alpha globulins bind certain
unidentified fluorcsccin-conjugatcd scrum proteins. In the second view, the alpha globulins,
without being retained, alter the nucleolus with
the result that in the prcscncc of calcium ions thc
conjugated globulins bind with the modilicd
nuclcoli. The first phase of the two-step rcaction
occurs in living cells cultured in vitro; this eliminates fixation artifact. The subsequcnt nucleolar
binding of conjugated globulins as a result of brief
incubation of cells with serum is an effect that
persists in the cclls and their progcny 1 wk after
the removal of serum (Fig. 2). Because of the
partial continuity of nucleolar protein from one
cell generation to the next (14), stable binding by
nucleoli of one or more serum globulins seems an
attractive intcrprctation. This vicw is also supported by the ability of cells incubated with serum
to reduce that scrum's titcr of nuclcolar reactivity.
On the other hand, it secms unusual that with
a tcnfold increase in cell number the serumincubated cells could absorb sufficient matcrial to
coat cvcry nuclcolus of every ccll in the 7th day
monolayer.
The nuclcolar-reactive component of serum
was associated consistently with the alpha globulins. At times, beta globulins or albumins appeared
to be present but, in purified preparations of these
substances, nuclcolar reactivity was not dcmonstrablc. On the other hand, one serum fraction
that modificd nucleoli under the conditions
described has bccn shown to bc of uniform electrophoretic mobility in the "slow" portion of the
alpha-1 globulin region. The molecular weight for
this species appears to be about one-sixth to oncfourth that of the alpha globulin average (6). By
weight, about one-sixth of this globulin is carbohydrate. Its nuclcolar affinity is removed by acid
or alkalinc hydrolysis, as well as by protcolytic
enzymes and pcriodatc action. Its idcntificd
sugars are sialic acid, hexosc, and hcxosamine, in
order of dccrcasing relative concentration. According to Winzlcr's definition (6), the nuclcolar rcac-
five material contains glycoprotein. The chemical
determination of sialic acid content of the fraction, associated with the loss of serum nucleolar
reactivity after virus action, suggests that a sialic
acid might be the reactive group split off by viral
neuraminidase (6).
Comparison of the nucleolar reactive fraction
with the better known glycoproteins reveals that
it contains less total carbohydrate and hexosamine
than the orosmucoid of Winzler (6). In addition,
our material is found in Cohn fraction III-1
instead of fraction VI, with the alpha globulins
instead of the prealbumins, and was not recovered
in the procedure used to prepare orosomucoid. A
sample of orosomucoid prepared by Dr. T. Inouye
from Cohn fraction VI (kindly supplied by Dr.
R. J. Winzler) was also found to lack nucleolar
reactivity. On the other hand, a relationship of
this component to fetuin is revealed in comparable
relative amounts of carbohydrate and individual
sugars, as well as its electrophoretic migration (6).
The nucleolar reactive material differs from fetuin,
however, in being absent from Cohn fractions IV
and VI. Fetuin has not been tested for nucleolar
reactivity. Purified fctuin lacks ability to support
the growth of primarily cultured mammalian
cells (15). With regard to chemical and nutritional properties, the nucleolar reactive material
is most like the alpha-1 glycoprotein of Lieberman
and Ore; however, with our material, the addition of peptones to the culture medium is not
required for cell growth (15).
The nature of the nucleolar component reacting with this glycoprotein-rich serum fraction is
not clear. Histones have been identified in animal
cell nucleoli (16) but, by definition, histones are
extractable with hypotonic NaC1 solutions or with
cold dilute mineral acids (10). On the basis of the
criteria given (10), the additional protein residual
in the nucleoli of extracted cells in Liau's experiments (16) was not a histone. In our experiment,
the solubility of the nucleolar protein in hypotonic
or hypertonic sodium chloride solutions and its
lack of solubility in 0.2 N HC1 pose a problem of
nomenclature. As a result of our observation that
nucleolar reaction with serum is independent of
nucleolar nucleic acids, we propose that residual,
histonelike (but nonhistone?) basic protein is
reacting with acidic globulins, possibly with sialic
acid-rich glycoproteins, or possibly with an unknown substance carried by these globulins. The
Published June 1, 1966
where serum proteins are required, their manner
of promoting growth remains unknown at the
molecular level (15, 17, 18, 19). T h e two lines of
reasoning might converge in a new concept for the
study of the nucleolar role in ribosome formation
(20). The events which must be considered are:
Does one or more serum proteins acting as a
"nuclear polyanion" (21) modify" nucleoli? Does
such modification result in increased R N A synthesis, reflected in an increased ribosome formation or
other aspects of cell growth? Finally, do the events
in mass cell cultures reflect in vivo phenomena?
The authors extend their gratitude to Dr. Gene
Stollerman, formerly Professor of Medicine at Northwestern University School of Medicine, for initial
guidance in this work. We thank Dr. Richard Winzler, formerly Professor of Physiological Chemistry
at the University of Illinois, for his constructive criticism as well as the analyses. Miss Betty Sullivan of
the Naval Medical Research Unit No. 4 prepared
the cell cultures. Vfe are indebted to Captain Lloyd
Miller, MC, USN, formerly Officer in Charge of the
Naval Medical Research Unit No. 4, for support of
this project. Doctors Albert Vatter, Smart Smith,
and Donald King, Jr., University of Colorado School
of Medicine, contributed greatly in the later stages
to our appreciation of the phenomenon investigated.
This work is Research Project M R 005.12-1102,
Bureau of Medicine and Surgery, Navy Department,
Washington, D. C. Completion of the project was
supported by United States Public Health Service
Pathology Training Grant No. 5 TL-GM-97702
awarded to Dr. Maisel.
The opinions and assertations contained herein
are those of the authors and are not to be construed
as official or reflecting the views of the Navy Department or of the Naval Service at large.
Received for publication 30 July 1965.
REFERENCES
1. MAISEL, J. C., Nucleolar reaction with normal
serum shown by nonimmune fluorescent
staining, J. Lab. and Clin. Med., 1962, 60, 357.
2. LIu, C., EATON, M. D., and HEYL, J. T., Studies
on primary atypical pneumonia. II. Observatioas concerning the development and immunological characteristics of antibody in patients, J. Exp. Med., 1959, 109,545.
3. KABAT, E. A., and MAYER, M. M., Experimental Immunochemistry, Springfield, Illinois,
Charles C Thomas, Publisher, 2nd edition,
1961, 162-164.
4. COHN, E. J., STRONG, L. E., HUGHES, W. L.,
472
MULFORD, D. J., ASHWORTH, J. N., MELIN,
M., AND TAYLOR,H. L., Preparation and properties of serum and plasma proteins: IV. A
system for the separation into fractions of the
protein and lipoprotein components of biological tissues and fluids, J. Am. Chem. Soe.
1946, 68,459.
5. SCHACHMAN, H. D., Ultraecntrifugation in Biochemistry, New York, Academic Press Inc.,
1959.
6. WINZLER, R. J., Glycoproteins, in The Plasma
Proteins, (F. W. Putnam, editor), New York,
Academic Press Inc., 1960, 309-347.
ThE ,]'OUtlNALOF CELIa BIOLOGY • VOLUME 29, 1966
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precise nature of the nucleolar protein or proteins
remains undetermined.
Associated with nucleolar reactivity is the
ability of several incompletely separated serum
fractions to substitute for whole serum in the
growth of cell monolayers from a primary explant
of trypsin-dispersed adult primate cells. This is also
true of one partly purified and fairly well characterized serum fraction. Such findings have no
exact counterpart in the tissue culture literature
(15, 18, 19). Nevertheless, it is premature to
describe the fraction in question as a proven
"growth factor." Quantitative studies of cell
culture growth kinetics, such as plating efficiency,
attainment of logarithmic growth, and average
generation time, have not been made in long term,
parallel comparisons with whole serum. Moreover, the two properties (nucleolar reactivity and
growth support) associated with one partly
characterized fraction may be mediated by different proteins. This possibility is suggested by
results obtained with the twenty initial run
curtain fractions (Table I I I , part B); growth
enhancement and nucleolar reactivity were not
always present together. However, in the early
studies of J a c q u e z and Barry (17), growthsupporting properties were found in the euglobulins and albumins; as mentioned above, alpha and
beta globulins and albumins have held the center
of interest as serum growth promoting fractions
(15). The few fractions of serum possessing both
nucleolar reactivity and growth promotion thus
present a problem of considerable interest.
Two lines of reasoning suggest the possibility of
a causal association between growth promotion
and nucleolar affinity. First, there is an acceleration of R N A synthesis in isolated rat liver nucleoli
divested of histones (16). Second, in the situations
Published June 1, 1966
15. TOZER, B. T., and PIRT, S. J., Suspension culture
of mammalian cells and macromoleeular
growth promoting fractions of calf serum,
Nature, 1964, 201, 375.
16. LIAU, M. C., HNILICA, L. S., and HURLBERT,
R. B., Regulation of R N A synthesis in isolated
nucleoli by histones and nucleolar proteins,
Proc. Nat. Acad. Sc., 1965, 53, 626.
17. JACO~UEZ, J. A., and BARRY, E., Tissue culture
media. The essential non-dialyzable factors
in h u m a n placental cord serum, or. Gen.
Physiol., 1951, 34, 765.
18. EVANS,V. J., BRYANT,J. C., KERR, H. A., and
SCHILLING, E. L., Chemically defined media
for cultivation of long term cell strains from
four mammalian species, Exp. Cell Research,
1964, 36, 439.
19. EAOLE, H., The sustained growth of h u m a n and
animal ceils in a protein-free environment,
Proc. Nat. Acad. So., 1960, 46, 427.
20. PERRY, R. P., Role of the nucleolus in ribonucleic acid metabolism and other cellular
processes, Nat. Cancer Inst. Monograph, 1964,
14, 73.
21. FRENSTER, S. H., Nuclear polyanions as derepressors of synthesis of ribonucleic acid,
Nature, 1965, 206, 680.
22. CANN, J. R., and KIRKWOOD, J. G., The fractionation of proteins by electrophoresis-convection, Cold Spring Harbor Symp. Quant. Biol.,
1950, 14, 9.
J. C. MAISEL ANn R. I. LYTLE A~nity of Cell Nucleolifor Serum
473
Downloaded from on June 17, 2017
7. DAvis, D. R., AND BUDD, R. E., Continuous
electrophoresis: quantitative fractionation of
serum proteins, or. Lab. and Clin. Med., 1959,
53,958.
8. BERTALANFFY, L., MASlN, F., and MASIN, M.,
The use of acridine-orange fluorescence
technique in exfoliative cytology, Science,
1956, 124, 1024.
9. DURYEE, W. R., Chromosomal physiology in relation to nuclear structure, Ann. New York
Acad. Sc., 1950, 50, 920.
10. DOUNCE, A. L., and SARKAR,N. K., Nucleoprotein organization in cell nuclei and its relationship to chromosome structure, in T h e
Cell Nucleus, (J. S. Mitchell, editor), New
York, Academic Press Inc., 1960, 206.
11. YOONGNER, J. F., Monolayer tissue cultures. I.
Preparation and standardization of suspensions of trypsin-dispersed monkey kidney cells,
Proc. Soc. Exp. Biol. and Med., 1954, 85, 202.
12. MORGAN,J. F., MORTON, H. G., and PARKER,
R. C., Nutrition of animal cells in tissue culture. I. Initial studies on a synthetic medium,
Proc. Soc. Exp. Biol. and Med., 1950, 73, 1.
13. DULBEGCO, R., and VOOT, M., Plaque formation
and isolation of pure lines with poliomyelitis
viruses, J. Exp. Med., 1954, 99, 167.
14. Hsu, T. C., ARRIGHI, F. E., KLEVECZ, R. R.,
and BRINKLEY, B. R., The nucleolus in mitotic
division of mammalian cells in vitro, J. Cell
Biol., 1965, 26, 539.